Custom DNA Oligos

Phosphorothioates

On this Page:
  Figure 1
  Synthesis
  Physical Properties
  Incorporation Guidelines
  Ordering Information
  Comments
  References


Many phosphate backbone variants have become popular recently in an attempt to overcome two challenges of the antisense approach to regulating gene expression: 1) the delivery of oligonucleotides to the interior of the cell through the highly impermeable barrier of the lipid bilayer that constitutes the cell's membrane, and 2) the equally important concern about the effective lifetime of the oligonucleotide in the exonuclease-rich environment of the cytoplasm.

Figure 1: Examples of phosphodiester and phosphorothioate internucleotide linkages.

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Phosphorothioates (or S-oligos) are a variant of normal DNA (See Figure 1) in which one of the nonbridging oxygens is replaced by a sulfur. The sulfurization of the internucleotide bond dramatically reduces the action of endo-and exonucleases2 including 5' to 3' and 3' to 5' DNA POL 1 exonuclease, nucleases S1 and P1, RNases, serum nucleases and snake venom phosphodiesterase. In addition, the potential for crossing the lipid bilayer increases. Because of these important improvements, phosphorothioates have found increasing application in cell regulation.

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Synthesis
Phosphorothioates are made by two principal routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by the more recent method of sulfurizing phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1, 2-bensodithiol-3-one 1, 1-dioxide (BDTD).4 The latter methods avoid the problem of elemental sulfur's insolubility in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield higher purity phosphorothioates.

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Physical Properties
A phosphorothioate linkage in inherently chiral. The sulfurization methods give a mixture of isomers at each incorporation site, resulting in 2n-1 isomers for an oligo of length n. Initially, there was much concern that this isomer mix would give unpredictable results. Fortunately, while short isomers are separable by HPLC and have a somewhat different resistance to nucleases, the isomer mix in any standard S-oligo preparation does not appear to affect the biological activity of the oligonucleotides. As mentioned above, S-oligos are quite amenable to purification. HPLC peaks will be somewhat broader due to both the isomer mix and the greater propensity of S-oligos for forming secondary structures. Longer elution times can be expected due to the increased hydrophobicity of these modified oligos. For this same reason one can expect that elution from reverse phase DNA purification cartridges will require a higher percentage of acetonitrile. The charge density of S-oligos is the same as a normal oligo and the change in mass is slight, so electrophoretic mobility is unchanged in denaturing PAGE. S-oligos do not kinase very well with 32-ATP and polynucleotide kinase. One can expect a decrease of labeling by a factor of 10. To eliminate this effect, incorporation of thioate linkages at the first three sites at the 5' end of the oligo should be avoided.

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Incorporation Guidelines
For oligonucleotides to resist degradation, phophorothioate linkages do not have to be present throughout the oligonucleotide. For example, RNase H is now thought to be a major partner in the antisense effect of oligonucleotides, and this enzyme requires a stretch of six unmodified phosphodiester linkages on the DNA strand to be effective. In cases where the major pathway of degradation is exonuclease attack, the entire oligo can be protected by the placement of a few thioate linkages at the ends of the oligo, thus preserving the RNase H activity as well as extending the half-life of the oligonucleotide. Additionally, extensively thiolated oligos are "sticky" and tend to bind to non-complementary sequences. This can give rise to spurious "antisense" effects from non-specific cell toxicity. Thus, fewer thioate incorporation can be beneficial in many instances.

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Ordering Information
Sigma offers high-purity phosphorothioate oligonucleotides at an exceptionally competitive price. We can also incorporate phosphorothioate linkages between specific residues, leaving the other linkages as normal phosphodiesters.

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Comments
If you find a need for phosphorothioates in your research, Sigma can provide you with fast delivery and high quality. If you have any questions regarding the preparation, purification or application of phosphorothioate oligos, or if you are just curious as to whether phosphorothioates can be of use in your application, please give us a call.

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References

  1. For a definitive bibliography of antisense research, see Chrisey, L.A. (1991) Antisense, Research and Development 1 (1), 65-113.
  2. Spitzer, S.; Eckstein, F. (1988) Nucl. Acids. Res. 16 (24), 11691-11704. Connolly, B.A.; Potter, B.V.L.; Eckstein, F.; Pingoud, A.; Grotjahn, L. (1984) Biochemistry 23, 3443-3453.
  3. Stein, C.A.; Pal, R.; Devico, A.L.; Hoke, G.; Mumbauer, S.; Kinstler, O.; Sarngadharan, M.G.; Letsinger, R.L. (1991) Biochemistry 30 (9), 2439-2444. Sayers, J.R.; Olsen, D.B.; Eckstein, F. (1989) Nucl. Acids Res. 17 (22) 9495. Manson, J.; Brown, T.; Duff, G. (1990) Lymphokine Research 9 (1), 35-42. Woolf, T.M.; Jennings, C.B.G.; Rebagliati, M.; Melton, D.A. (1990) Nucl. Acids Res. 18 (7), 1763-1769. Leiter, J. M.E.; Agrawal, S.; Palese, P.; Zamecnik, P.C. (1990) Proc. Natl. Acad. Sci. USA 87 (9), 3430-3434. Reed, J.C.; Stein, C.; Subasinghe, C.; Haldar, S.; Croce, C.M.; Yum, S.; Cohen, J. (1990) Cancer Research 50 (20), 6565-6570.
  4. Iyer, R.P.; Egan, W.; Regan, J.B.; Beaucage, S.L. (1990) J. Am. Chem Soc. 112, 1254-1255. Iyer, R.P.; Phillips, L.R.; Egan, W.; Regan, J.B.; Beaucage, S.L. (1990) J. Org. Chem. 55, 4693-4699.
  5. Dagle, J.M.; Weeks, D.L.; Walder, J.A. (1991) Antisense, Research and Development 1 (1), 11-20. Maher, L.J.; Dolnick, B.J. (1988) Nucl. Acids Res. 16, 3341-3358. Walkder, R.Y.; Walkder, J.A. (1988) Proc. Natl. Acad. Sci. USA 85, 5011-5015.

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